MBE Advance Access originally published online on January 24, 2008
Molecular Biology and Evolution 2008 25(3):487-496; doi:10.1093/molbev/msm245
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Research Articles |
Mitochondrial Genome of the Colorless Green Alga Polytomella capuana: A Linear Molecule with an Unprecedented GC Content
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada
E-mail: robert.lee{at}dal.ca.
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Abstract |
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One common observation concerning mitochondrial genomes is that they have a low guanine and cytosine content (GC content); of the complete mitochondrial genome sequences currently available at the National Center for Biotechnology Information (NCBI) (July 2007), the GC content ranges from 13.3% to 53.2% and has an average value of 38%. Here, we present the GC-rich mitochondrial genome (57% GC) of the colorless green alga Polytomella capuana. The disproportion of GC among the different regions of the P. capuana mitochondrial DNA (mtDNA) suggests that a neutral process is responsible for the GC bias. We propose that a biased gene conversion mechanism resulted in the GC-rich state of the P. capuana mtDNA. In addition, our analysis indicates that the P. capuana mitochondrial genome is a single 13-kb linear molecule with telomeres, which have a closed (hairpin-loop) conformation: a novel terminal structure among described linear green-algal mtDNAs. Furthermore, using a series of GC-rich inverted repeats found within the P. capuana mitochondrial genome, we describe recombination-based scenarios of how intact linear mtDNA conformations can be converted into the fragmented forms found in other Polytomella taxa.
Key Words: Polytomella capuana • green algae • mitochondrial DNA (mtDNA) • GC content • biased gene conversion • telomeres
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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One of the most distinguishing characteristics of mitochondrial genomes as compared with nuclear genomes is their low GC content; of the 1,125 complete mitochondrial DNA (mtDNA) sequences available at NCBI as of July 2007, the GC content ranges from 13.3% to 53.2% and has an average value of 38%. Although sampling is highly biased toward animal mitochondrial genomes (1,015 out of the 1,125), the trend of having a low GC content is seen throughout other major groups, including fungi (17.1–43.2% GC), the Archaeplastida (Adl et al. 2005
) (22.2–45.2% GC), and the heterogeneous group of eukaryotic unicells negatively defined as not belonging to animals, fungi, or the Archaeplastida (14.0–41.2% GC). Various hypotheses for explaining why mtDNA is GC poor have been proposed. For example, it has been suggested that the low levels of G and C are due to a mutational bias caused by the loss of DNA repair genes in the endosymbiotic genome that gave rise to the mitochondrial genome (Glass et al. 2000; Moran 2002; Burger and Lang 2003). Others contend that a low GC content correlates with adaptation to an intracellular lifestyle where high levels of ATP and UTP relative to GTP and CTP make replication and transcription of an AT-rich genome more efficient (Howe et al. 2002; Rocha and Danchin 2002). Convergent evolution toward a low GC content is also seen in the genomes of chloroplasts, symbionts, and endocellular parasites (Dybvig and Voelker 1996; Ogata et al. 2001; Kusumi and Tachida 2006).
The mitochondrial genome of the colorless green algae Polytomella capuana attracted our interest because of its potential for having a particularly high GC content relative to other available mitochondrial genome sequences, not only for green algae but also for eukaryotes in general. This view was motivated by the high GC content of a 768-nt segment of cox1 from the mtDNA of P. capuana (GenBank accession number DQ221113; Mallet and Lee 2006), especially at sites expected to be under low selective constraint, such as 4-fold degenerate sites, which are third-position codon sites that can tolerate any of the 4 nt without altering the amino acid specified.
Polytomella is a group of wall-less and colorless unicells (Pringsheim 1955) belonging to the "Reinhardtii clade" (Gerloff-Elias et al. 2005) of chlorophycean green algae. Available Polytomella taxa fall into 3 lineages (Mallet and Lee 2006). Two of these lineages, represented by Polytomella parva and Polytomella strain 63-10 (Sammlung von Algenkulturen Göttingen [SAG]), possess fragmented mitochondrial genomes (Fan and Lee 2002; Mallet and Lee 2006). Whereas the third and earliest branching lineage, represented by P. capuana, appears to have an intact linear mitochondrial genome (Mallet and Lee 2006). Substantial sequence data exist only for the mitochondrial genome of P. parva, which is comprised of 2 linear fragments with estimated sizes of 13.5 and 3.5 kb (Fan and Lee 2002). Sequence information spanning 97% and 86%, of the 13.5- and 3.5-kb fragments, respectively, is available. The 3.5-kb fragment encodes only 1 gene (nad6), which is missing from the 13.5-kb fragment. The telomeres of both fragments contain virtually identical inverted repeats that are at least 1.3 kb in length; however, the extreme termini of both fragments still remain to be sequenced.
Chlamydomonas reinhardtii, a close relative to Polytomella taxa (Nakayama et al. 1996; Pröschold et al. 2001; Gerloff-Elias et al. 2005), has a completely sequenced linear mtDNA molecule of 15.8 kb with terminal inverted repeats of 531 or 532 nt including a 39- to 41-nt noncomplementary 3' extension (Gray and Boer 1988; Michaelis et al. 1990; Vahrenholz et al. 1993). Based on the potential interaction of the telomeres with internal repeats, 2 models of replication have been proposed for the C. reinhardtii mtDNA (Vahrenholz et al. 1993). There are no apparent similarities between the telomeric sequences of P. parva and C. reinhardtii (Fan and Lee 2002), and a model describing how the P. parva mtDNA may replicate has not yet been proposed.
Taken as a whole, sequence data from the P. capuana mtDNA will provide useful information on nucleotide composition biases in mitochondrial genomes as well as knowledge about the evolution of mitochondrial telomeres and the mechanisms through which intact linear mtDNA conformations were converted into fragmented forms. With these motives in mind, we sequenced to completion the P. capuana mitochondrial genome.
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Materials and Methods |
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Strain, Culture Conditions, Mitochondrial Enrichment, and DNA Extraction
We used a stock of P. capuana (SAG strain 63-5) made axenic by Mallet and Lee (2006)
. Cells were cultured at 22 °C in Polytomella medium (0.1% tryptone, 0.2% yeast extract, and 0.2% sodium acetate) and harvested in the late logarithmic phase of growth (OD750 nm = 0.4; determined with a Bausch and Lomb Spectronic 20 spectrophotometer) by centrifugation (1,000 x g) at 4 °C. Cells were disrupted with a Dounce homogenizer. The mitochondrial-enriched fraction was prepared and treated with DNase I following procedure B of Ryan et al. (1978). Isolation of DNA followed the method of Ryan et al. (1978), with the exception that there was no further DNA purification step employing preparative CsCl gradient centrifugation.
DNA Amplification
Polymerase chain reaction (PCR) experiments were performed in High Fidelity Platinum SuperMix (Invitrogen, Carlsbad, CA) using DNA from a mitochondrial-enriched fraction as the template. DNA was initially denatured at 94 °C for 3 min, then amplified by 35 cycles of denaturation at 94 °C for 45 s, annealing at 50–60 °C (depending on the melting temperature of the primers), and extension at 72 °C; there was a final extension at 72 °C for 10 min. The telomeric regions of the P. capuana mtDNA were amplified using: 1) the long-walk PCR method of Katz et al. (2000), 2) terminal deoxynucleotidyl transferase (TdT) tailing as described by Förstemann et al. (2000) and Bah et al. (2004), and 3) standard PCR amplification (as described above).
DNA Blotting and Hybridization
Blotting of agarose gels onto Hybond-N+ membranes (Amersham, Buckinghamshire, UK) was performed using the Vacublot XL system (Amersham). Probes used in this study were labeled and hybridized to samples with the AlkPhos Direct Labelling and Detection System (Amersham) following the manufacturer's instructions. Label was detected by exposing the membranes to Fuji Super RX medical X-ray film.
Cloning and Sequencing of DNA Fragments
PCR-, long-walk PCR-, and TdT-tailing products were separated by agarose gel electrophoresis, purified with the QIAquick Gel Extraction Kit (Qiagen, Germantown, MD), and then cloned using the TOPO TA Cloning Kit (Invitrogen). Plasmid DNA was extracted with the QIAquick Spin Miniprep Kit (Qiagen). PCR products and the clones derived from PCR products were sequenced on both strands at the Centre for Applied Genomics, Hospital for Sick Children, Toronto, Canada.
Sequence Analysis
Sequences were edited and assembled using CodonCode Aligner (Version 1.5.2). The Blast network services (Altschul et al. 1990) were employed for sequence similarity searches. Protein-coding regions not initially detected in the Blast search were identified with the Open Reading Frame Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The boundaries of the mitochondrial rRNA-coding modules were estimated by sequence comparisons with their counterparts in P. parva and C. reinhardtii mtDNA. Genes encoding tRNAs were located using tRNAScan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/; Lowe and Eddy 1997). Repeated regions were identified using Repeat Finder (http://www.proweb.org/proweb/Tools/selfblast.html) and REPuter (Kurtz et al. 2001). Secondary structures and folding energies of the inverted repeat sequences were predicted with Mfold (Zucker 2003). The equation used for calculating the GC-skew value was (G – C)/(G + C) and that for the AT skew was (A – T)/(A + T). The cumulative GC-skew plot was formulated using the GenSkew software program (http://mips.gsf.de/services/analysis/genskew).
Nucleotide Sequence Accession Number
The complete sequence of the P. capuana mtDNA is deposited in GenBank under the accession number EF645804.
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General Features
The P. capuana mitochondrial genome is a single linear molecule of 12,998 nt with terminal inverted repeats (i.e., the sequence of one terminus is present in an inverted orientation relative to the other terminus) (fig. 1). The size and conformation of the P. capuana mtDNA were confirmed by pulse-field gel electrophoresis and restriction endonuclease digestion patterns (data not shown). The coding regions in this mtDNA are arranged into 2 unequally sized clusters with opposite transcriptional polarities, which proceed outwards toward the ends of the genome. The region separating the 2 transcriptional orientations shows no similarity to the potential promoter sequence identified in the corresponding region of the P. parva (Fan and Lee 2002
) and C. reinhardtii mtDNAs (Duby et al. 2001). The complement and arrangement of genes in the P. capuana mtDNA parallels that of P. parva, except for nad6, which is located internal to the left terminal repeat in P. capuana (fig. 1) but on a separate chromosome in P. parva (fig. 1). The sole tRNA gene, trnM(cau), has characteristics that are consistent with a role in elongation rather than initiation, like the mitochondrial-encoded trnM(cau) of other Reinhardtii-clade taxa (Boer and Gray 1988; Denovan-Wright et al. 1998).
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Of the 12,998 nt comprising the P. capuana mtDNA, 10,662 nt (82%) code for proteins and functional RNAs, and 2,336 nt (18%) represent noncoding DNA. The latter can be subdivided into intergenic regions and terminal repeats, which constitute 555 nt and 1,780 nt (890 nt at each terminus), respectively. The 19 intergenic regions identified in the P. capuana mtDNA range in size from 3 to 62 nt and have an average length of 29 nt.
The P. capuana has a genome-wide GC bias; the overall GC content is 57.7%. The allocation of G versus C (GC skew) on the main sense strand (the strand encoding the gene for cox1) is negligible with a value of only 0.006. The distribution of A versus T is slightly more skewed at –0.09, reflecting a slight tendency toward T on the main sense strand.
GC Bias by Region
The GC content of the P. capuana mtDNA differs considerably among the various regions of the genome. Table 1 shows that the GC values in P. capuana mtDNA exceed those of P. parva and C. reinhardtii for all defined regions.
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Base composition of the coding mtDNA in P. capuana has an average GC content of 56.4% (table 1). This value reflects both rRNA- and protein-coding regions evenly. In opposition, the trnM gene is slightly GC poor (47.9%). The inflated GC content of the protein-coding genes in the P. capuana mtDNA comes from a large number of codons ending in G or C (76%) (table 1); when considering only 4-fold degenerate sites, this value is even higher (85%). In both of these cases, the occurrence of G versus C is approximately equal. At the more functionally constrained first and second codon positions, the base compositions are less GC rich, with values of 52% and 41%, respectively (table 1). The protein-coding genes in the mitochondrial genomes of P. parva and C. reinhardtii show a tendency toward A and T at all 3 codon positions (table 1). The P. capuana protein-coding genes, in spite of being GC rich, show a derived amino acid composition similar to that of the P. parva and C. reinhardtii protein-coding genes (supplementary fig. 1, Supplementary Material online). Alanine is the only significant exception to this trend, encoded 308 times in the P. capuana mtDNA but only 257 and 215 times in that of C. reinhardtii and P. parva, respectively. It is noteworthy that alanine is encoded by the GC-rich codon family GCN.
Noncoding DNA in the P. capuana mitochondrial genome is more GC rich than the coding DNA (table 1); the average GC content of the telomeres is 58.7% and that of the intergenic spacer regions is 68% (table 1), with the individual intergenic regions ranging from 55% to 100%. Noncoding DNA in both the P. parva and C. reinhardtii mitochondrial genomes is GC poor (<50%), except for the telomeres of C. reinhardtii, which are slightly GC rich (54%) (table 1).
Repetitive Elements
Repetitive elements in the P. capuana mtDNA can be divided into 2 categories: short inverted repeats, which can be folded into stem-loop structures and long-terminal inverted repeats, which make up the telomeric regions.
Short Inverted Repeat Elements
Sixteen pairs of inverted repeats were identified throughout the coding and noncoding regions of the P. capuana mtDNA (fig. 2). The inverted repeats vary in length from 5 to 27 nt, and all 16 pairs can be folded into stem-loop (hairpin) structures (fig. 2). Although there is no sequence identity between pairs of inverted repeats and though the size of stems and loops among their different predicted secondary structures varies considerably, 2 trends are apparent: 1) the stems are GC rich (>60%), whereas the loops are less so (
50%); and 2) the location of the stem-loop structures correlate with the start and end of coding regions.
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Twelve of the inverted repeat pairs have an arrangement where one inverted repeat is found in intergenic DNA and the other (matching) inverted repeat is located in an adjacent coding region, thereby resulting in a stem-loop structure that spans both coding and noncoding DNA. In 9 of 12 cases where this occurs, the "loop" portion of the hairpin contains the start of a coding region and the "stem" component is adjacent to the end of a coding region (fig. 2). Every protein-coding gene and all but 2 of the rRNA-coding modules found in the P. capuana mtDNA are bordered by potential stem-loop structures.
A few of the inverted repeats share sequence identity with other parts of the genome. For example, a 20-nt perfect-match sequence corresponding to the stem of the cox1/nad4 hairpin structure (fig. 2) was found inserted into nad6. Similarly, a 12-nt portion of the cob/nad6 stem (fig. 2) was found inserted into nad5. The location of the inverted repeat between the nad6 and cob genes (comprising one-half of the stem in the cob/nad6 stem-loop structure) corresponds to the region that is fragmented in other Polytomella lineages.
The "global minimum" of a cumulative GC-skew plot (a plot that measures the change in G vs. C over a moving window; supplementary fig. 2, Supplementary Material online) of the main sense strand in the P. capuana mtDNA occurs at the apex (nucleotide 9,934) of the largest stem-loop structure in the genome—that between the regions encoding the rrnl-L1 and rrns-S4 gene fragments (fig. 2). The global minimum of a GC-skew plot is often used to predict the origin of replication in bacterial and mitochondrial genomes (Grigoriev 1998). A similar but much weaker stem-loop structure was found in the mitochondrial genome of P. parva at the corresponding region.
Telomeric Repeats
The terminal regions (telomeres) of the P. capuana mitochondrial genome proved unamenable to standard cloning techniques. Sequencing of the telomeres was thus achieved using terminal TdT-tailing (Förstemann et al. 2000; Bah et al. 2004) and long-walk PCR methods (Katz et al. 2000). The nature of these protocols is that the TdT-tailing method allows access to the 3' end of a telomere, whereas the long-walk PCR approach works outwards on the strand containing the 5' end; by using each of these techniques, one can sequence both strands of a telomere independently.
Sequencing results from TdT-tailing and long-walk PCR (fig. 3) suggest that the terminal regions of the P. capuana mtDNA exist (in vitro) in 2 separate conformations: a closed (hairpin-loop) conformation and an open (nicked-loop) conformation. The nucleotide sequence from both these conformations appears to be identical; however, that from the closed conformation seems to terminate with a 220-nt single-stranded loop, whereas in the open conformation this loop appears nicked (fig. 3). The location of the nick was shown to vary but was most often observed at the apex of the loop. Further experiments using standard PCR techniques were performed to confirm the sequence of the telomeric regions. As expected, PCR reactions were able to proceed through the terminal loop structure. Gel analyses and restriction digest results also support the idea that the P. capuana mtDNA telomeres exists in both an open and a closed conformation (supplementary figs. 3 and 4, Supplementary Material online).
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Discussion |
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Nucleotide Composition Bias
The P. capuana mtDNA has the highest GC content (57.7%) of any completely sequenced mitochondrial (or organelle) genome currently deposited in the NCBI organelle genome data bank (http://www.ncbi.nlm.nih.gov/genomes/static/euk_o.html; July 2007). Heretofore, all completely sequenced green-algal mitochondrial genomes have had a GC content below 46%, which leads to the question: why is the mtDNA of P. capuana GC rich? Or alternatively, why is it so AT poor? Hypotheses about biased nucleotide composition fall into 2 categories, which we will call: selectionist based and neutralist based. The former suggests that nucleotide bias is the result of natural selection; for example, this approach is often used to argue that GC richness is an adaptation to homeothermy (Jabbari and Bernardi 2004
) or UV tolerance (Singer and Ames 1970; Kellogg and Paul 2002). In opposition, neutralist-based hypotheses posit that nucleotide inequities arise by either biased mutation pressure or biased gene conversion (BGC). Biased mutational pressure is often applied to the low GC content of organelle and endocellular parasite genomes (Dybvig and Voelker 1996; Ogata et al. 2001; Kusumi and Tachida 2006), whereas BGC, which results from biased repair of mismatches in heteroduplexed recombination intermediates (Holmquist 1992; Eyre-Walker 1993; Galtier et al. 2001; Galtier and Duret 2007), has been correlated to GC richness. Furthermore, gene conversion, which is believed to occur in organelle genomes (Birky and Walsh 1992; Walsh 1992; Khakhlova and Bock 2006; Tatarenkov and Avise 2007), may be GC biased in mitochondrial systems: a positive correlation between the GC content of repeat sequences and their recombinogenic properties in mitochondrial genomes has been observed in fungi and plants (Dieckmann and Gandy 1987; Clark-Walker 1989; Weiller et al. 1989; Nakazono et al. 1995), and has been suggested for certain Reinhardtii-clade green algae (Boer and Gray 1991; Nedelcu 1997, 1998; Nedelcu and Lee 1998).
Within the P. capuana mtDNA, the GC bias is highest at what are typically regarded as among the most neutrally evolving positions (intergenic and 4-fold degenerate sites) and it is lowest at what are generally considered more functionally constrained positions (first and second codon sites). This disproportion of GC content among the different regions of the P. capuana mitochondrial genome is best explained by the negative selection principle of the neutral theory of molecular evolution (Kimura 1983). If a neutral process is responsible for the GC bias of P. capuana mtDNA, is that process the effect of biased mutation pressure or BGCGC?—or both? Any attempt to choose between these possibilities is purely speculative; however, we tend to favor a predominant role of BGCGC, for reasons discussed below.
Duret et al. (2006) proposed that in the nuclear DNA of mammals the GC content of a given region reflects a balance between an AT-biased mutation process and BGCGC (a GC-biased fixation process). They further suggest (following the theory of Galtier 2004) that GC-rich regions form rapidly during times when the recombination rate is high enough for BGCGC to be effective; then, once the recombination rate decreases, the GC content declines slowly as a result of AT-mutation pressure. If we suppose that mitochondrial genomes have an AT-biased mutation process and that gene conversion in mtDNA is GC biased, we can apply the model of Duret et al. to mitochondrial systems. Under this premise, the history of most mitochondrial genomes would reflect an AT-biased mutation process and a recombination rate where BGCGC is ineffective, thereby giving rise to AT-rich mitochondrial genomes. For the mtDNA of P. capuana, however, we suggest a recent history with high levels of recombination, thus shifting the nucleotide composition toward G and C. According to this hypothesis, any new mutation in one of the multiple copies of the P. capuana mtDNA that results in heteroplasmy, where one allele is A•T at a given site and the other allele is G•C at the same position, should be preferentially converted to the G•C allele via BGCGC. Reasons why the P. capuana mtDNA may have undergone an increased rate of recombination would have to be entirely suppositional at this time. One intriguing observation, however, is that the isolate of P. capuana used for this research came from Italy (Capua), whereas the available mtDNA sequences of P. parva came from a strain isolated in the United Kingdom (Cambridge). Sun exposure between these 2 regions differs substantially, invoking the possibility that an elevated recombination rate may be a repair response to UV damage in the P. capuana mtDNA; but because little is known about the full habitat range of either P. capuana or P. parva, little weight can be placed on this observation. Moreover, we have been unable to get P. capuana to form cysts in the laboratory, unlike the other known species of Polytomella, which may make it more susceptible to UV damage.
Linear Mitochondrial Genomes
The P. capuana mitochondrial genome is one of several examples of linear-type mtDNA from the Reinhardtii clade of chlorophycean green algae (for a compilation see Laflamme and Lee 2003; Mallet and Lee 2006; Popescu and Lee 2006). When comparing the telomeres of the P. capuana mtDNA with those of other Reinhardtii-clade algae for which telomere data are available (namely C. reinhardtii and P. parva), no universal themes are apparent. Both the length and sequence of the telomeres in the P. capuana mtDNA differ substantially from those of P. parva and C. reinhardtii. Furthermore, the terminal structures of the P. capuana mtDNA, which appear to exist in either closed (hairpin-loop) or open (nicked-loop) conformations, are different from those of C. reinhardtii, which are made up of 3' single-stranded extensions (Vahrenholz et al. 1993). We are unable to exclude the possibility, however, that the open telomeric conformation is the result of nicking during the DNA extraction process and does not normally exist in vivo. Polytomella capuana is not the first example of a linear mtDNA with terminal hairpins; they are found in the linear mitochondrial genome of the yeast Pichia (Dinouël et al. 1993), at one end of the Paramecium mtDNA (Pritchard and Cummings 1981), and also in the mitochondrial plasmid of the plant pathogenic fungus Rhizoctonia solani (Miyashita et al. 1990). Other examples of this telomeric structure come from viruses of eukaryotic cells (Baroudy et al. 1982; González et al. 1986), including a virus which infects certain species of the green-algal genus Chlorella (Rohozinski et al. 1989), and from the bacterial plasmids of the genus Borrelia (Hinnebusch and Barbour 1991). Although the terminal structures of the P. parva mitochondrial genome are not characterized, preliminary gel electrophoresis results (Smith and Lee, unpublished data) suggest that they are similar in conformation to those of P. capuana.
All linear genomes must develop a strategy to overcome the end replication problem, as defined by Olovnikov (1971) and Watson (1972). For the C. reinhardtii mitochondrial genome, 2 replication models have been proposed (Vahrenholz et al. 1993). One model involves reverse transcription of an internal repeat via a putative mitochondrial–encoded reverse transcriptase (RTL), whereas the second model takes into account that the reverse transcriptase gene may be nonfunctional. No open reading frames resembling a reverse transcriptase-like gene were found in the P. capuana (or P. parva) mitochondrial genome, and the fact that the structure of its telomeres depart from that of C. reinhardtii suggest that it uses a different replication strategy. Although in the case of P. capuana no strategy is apparent, many replication models for linear genomes with terminal hairpins have been suggested (Cavalier-Smith 1974; Bateman 1975; Pritchard and Cummings 1981; Baroudy et al. 1983; Dinouël et al. 1993; Traktman 1996).
Origin of Fragmentation
Although linear-fragmented mitochondrial genomes have been observed in other eukaryotic lineages, including 4 classes of Cnidaria (Warrior and Gall 1985; Bridge et al. 1992; Ender and Schierwater 2003) and the ichthyosporean Amoebidium parasiticum (Burger et al. 2003), the Polytomella genus represents a unique example in that substantial mtDNA sequence data exist for both a linear-fragmented and a linear-intact genome from 2 closely related taxa, thus allowing for comparative analyses.
Assuming that the linear bipartite mitochondrial genome of P. parva and Polytomella strain 63-10 were derived from an ancestral unfragmented linear molecule, we can posit that the ancestral mtDNA conformation may have been similar to that of P. capuana: a single linear chromosome with terminal inverted repeats. Furthermore, because the gene arrangement of the unfragmented P. capuana mitochondrial genome is parallel to that of the fragmented P. parva mitochondrial genome—fragmentation notwithstanding—we can consider the ancestral Polytomella gene arrangement to be equivalent to that of P. capuana, where the gene encoding nad6 is found internal to the left telomere. Under these premises, the P. capuana mitochondrial genome can act as a model for understanding fragmentation of the ancestral Polytomella mtDNA.
Several features of the P. capuana mtDNA suggest that the nad6 gene is in an unstable region. Its terminal position lends itself to recombination and possible fragmentation more readily than other internally located genes: recombination rates have been shown to be higher at the termini of linear chromosomes as compared with their more centrally located regions (Eichler and Sankoff 2003; See et al. 2006). Also, the intergenic sequence between the nad6 and cob genes is comprised of a potentially unstable GC-rich inverted repeat: in the mitochondrial genomes of Neurospora cerevisiae and Saccharomyces cerevisiae GC-rich inverted repeat sequences were shown to have inflated rates of recombination causing genome rearrangements or deletion mutations that were maintained in the population of mtDNAs (Almasan and Mishra 1988; Clark-Walker 1989). In fact, portions of the P. capuana nad6/cob intergenic sequence were found inserted into both the gene for nad5 and the telomeres, indicating that this sequence may have mobile properties, perhaps similar to those of the GC clusters found in the mtDNA of certain Saccharomyces species (de Zamaroczy and Bernardi 1986). By using a portion of the short inverted repeat sequences in the nad6/cob intergenic region and homologous sequences in the telomere regions, we were able to outline a scenario 1 and 2 involving illegitimate recombination between P. capuana mitochondrial genomes, which produce products structurally similar to the 3.5-kb and 13.5-kb mtDNAs of P. parva, respectively (fig. 4A). Lineages with such fragmented mtDNA forms may have become fixed by random genetic drift especially if Polytomella populations went through a bottleneck. According to this possibility, one might expect an ongoing, low-level production of such fragmented mtDNA forms from the intact mtDNA structure in P. capuana. Although we were not able to detect these forms by a PCR approach, we were able to reliably detect PCR products, using a wide range of primer combinations, that are consistent with other illegitimate recombination events involving the short inverted repeat sequences in the telomere regions as shown in figure 4B.
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Short Inverted Repeats
The short GC-rich inverted repeat sequences in the P. capuana mtDNA evoke several questions regarding their evolution and function, such as: 1) do they play a role in gene expression, 2) do they have mobile properties, and 3) are they related to the inverted repeats found in other mitochondrial genomes. Inverted repeat sequences capable of forming stem-loop structures have been described in the mitochondrial genomes of animals, fungi, plants, and a series of Reinhardtii-clade green algae including C. reinhardtii (Boer and Gray 1986
, 1991) and 2 species of Volvox (Aono et al. 2002). In the above cases, the inverted repeat sequences are generally restricted to intronic or intergenic regions, and in many cases, they have been implicated in mobility or RNA processing (Boer and Gray 1986, 1991; Nedelcu and Lee 1998; Aono et al. 2002). What distinguishes the inverted repeats of the P. capuana mtDNA from those of most other mitochondrial systems is that they span both coding and noncoding DNA, often resulting in stem-loop structures that contain the start or the end of a gene; this arrangement suggests that the inverted repeats and their putative secondary structures may have a role in gene expression, perhaps akin to mammalian mitochondrial systems, where large polycistronic transcripts are processed by cleavage at the boundaries of tRNA sequences, which flank almost every gene (Clayton 1984). We are aware of only one other mitochondrial genome with a similar orientation of inverted repeats: the dinoflagellate Amphidinium carterae (Nash et al. 2007); however, the utility of the repeats within this taxon are also unknown. The absence of inverted repeat sequences from the P. parva mitochondrial genome indicates that the inverted repeats in the P. capuana mtDNA may be invasive elements, conceivably appearing in P. capuana after its divergence from P. parva; but we can not eliminate the possibility that the elements were lost from the P. parva mtDNA. Two of the inverted repeats share sequence identity with other parts of the genome; 20 nt of the cox1/nad4 hairpin structure was found inserted into nad6, and 12 nt of the cob/nad6 stem was inserted into nad5. This suggests that some of the inverted repeat sequences in the P. capuana mitochondrial genome may have mobile properties, but the lack of conservation in primary sequence among the different inverted repeats implies that if there is mobility it may be dependent on secondary structure rather than a specific nucleic acid sequence. |
Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary figures 1–4 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We are indebted to Tudor Borza for his expert advice throughout the experimental and writing phases of this work. We also thank Gertraud Burger and Emmet O'Brien for graciously providing the nucleotide composition data of the currently available mitochondrial genome sequences. This work was supported by a grant to R.W.L. from the Natural Sciences and Engineering Research Council (NSERC) of Canada. D.R.S. received a Canada Graduate Scholarship from NSERC.
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Footnotes |
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Charles Delwiche, Associate Editor
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